DESCRIPTION (adapted from the application) The long-term goal of this research is to understand the role of temperate phages in, and the contribution of phage-encoded gene products to, the virulence of enterohemorrhagic Escherichia coli (EHEC). EHEC are emerging foodborne pathogens that have caused large-scale outbreaks of gastrointestinal illness in developed countries during the past two decades. Intestinal infection can lead to diarrhea, hemorrhagic colitis or more severe systemic complications, such as hemolytic uremic syndrome. The production of Shiga toxins by EHEC strains plays an important role in the development of serious complications following EHEC infection. Two immunologically distinct Shiga toxins, designated Stx1 and Stx2, have been identified among clinical E. coli isolates of many serotypes. Genes for both Shiga toxin types in E. coli have been shown to be encoded on lysogenic lambdoid bacteriophages. These Shiga toxin-encoding phages have played an important role in transmitting the stx genes during the evolution of Stx-producing enteric pathogens, and continue to be involved in ongoing dissemination of Shiga toxin genes to new hosts. In addition, recent studies have shown that the toxin genes appear to be integrated into the lytic circuitry of these phages in such a way that prophage induction leads to increased toxin gene expression and concomitant release of toxin by host cell lysis. One aim of the experiments outlined in this application is to define more clearly the roles of phage-encoded functions in toxin gene expression and toxin release. A second aim of the application is to investigate other phage-encoded genes that are postulated to play roles in lysogenic conversion. The products of these genes may affect processes, such as colonization or immune evasion, that could contribute to the virulence of lysogenic bacteria. Using the Stx2-encoding phage 933W, which has been sequenced in its entirety, directed mutations in individual genes will be constructed. The effects of these mutations on Shiga toxin production or other interactions with host cells will be assessed in vitro, and effects on virulence using a mouse model system will be determined.